While it is now appreciated that tumor cells contain thousands of mutations, identifying their source(s) has remained problematic. Whole genome sequencing from colorectal and endometrial cancers recently identified mutations in DNA polymerase (Pol) epsilon, one of three main eukaryotic nuclear replication Pols. These mutations largely reside in the exonuclease domain, are associated with a hypermutated phenotype, and are predicted to promote tumorigenesis. However, the effects of these somatic mutations on enzyme function have not yet been studied. To address this, we introduced several of these mutations in recombinant human Pol ε and measured their effects on replication fidelity in vitro. While the mutants retained wild type DNA synthesis activity, 3'‐5' exonuclease proofreading was suppressed. Surprisingly, this suppression was variable, ranging from ~2‐fold to almost complete ablation, and resulted in increases in frameshifts and base pair substitutions in an in vitro forward mutation assay. Mutants also had increased ability to bypass abasic sites. Exonuclease inactivation of one POLE allele in human cells resulted in decreased sensitivity to H2O2 treatment. These results suggest that Pol ε mutant alleles may promote viability and mutagenesis, and possibly tumorigenesis, in part through bypass of oxidative stress‐induced DNA lesions. Grant Funding Source: NIH RR020152
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